All-trans retinal is generated in photoreceptors as a result of the detection of light by the visual pigment. All-trans retinal is a cytotoxic substance that can initiate oxidative damage and is normally removed rapidly from the photoreceptor. Progress in the previous funding period showed that efficient clearance of all-trans retinal requires metabolic energy and the proper functioning of retinoid processing pathways. The long-term goal of this project is to determine the intracellular, extracellular, and environmental factors that influence clearance of all-trans retinal and protect photoreceptors from its toxicity. The accumulation of all-trans retinal and the resultant oxidative damage will be investigated in isolated photoreceptor membranes, and in living frog and mouse photoreceptors. To carry out these studies, we will combine biochemical assays of all-trans retinal and oxidative damage with fluorescence imaging and oxidation of single photoreceptors using one- and two-photon microscopy. The aims of the proposed research are to: 1. Determine whether the buildup of all-trans retinal promotes oxidative damage. 2. Determine whether improved removal of all-trans retinal prevents oxidative damage. Defects in all-trans retinal removal have been implicated in a variety of retinal diseases, ranging from impaired dark adaptation to complete blindness. The cytotoxicity of all-trans retinal could be a factor in a broad spectrum of pathological conditions, including Age-Related Macular Degeneration, the leading cause of blindness in the US, metabolic diseases like diabetes, and damage to the retina inflicted by sunlight or by ophthalmic instruments. Impaired removal of all-trans retinal may play a role in Stargardt's disease, an early onset form of macular degeneration, and Oguchi's disease, a form of stationary night blindness. The proposed research will determine the basic parameters underlying the photo-oxidative potential of all- trans retinal in photoreceptors. It will determine the metabolic limits at which the clearance of all-trans retinal becomes compromised and allow an assessment of the impact of metabolic perturbations on the long-term health of photoreceptors. It will clarify the role of potential risk factors for the development of Age-Related Macular Degeneration, and elucidate the relation between metabolic limitations, light exposure, and disruptions in retinal processing in Stargardt's and Oguchi's diseases. PUBLIC HEALTH RELEVANCE: All-trans retinal is continuously generated in photoreceptors as a result of the detection of light by the visual pigment. All-trans retinal is a cytotoxic substance that has been implicated in a variety of diseases of the retina, including Age-Related Macular Degeneration, Stargardt's, and some forms of Retinitis Pigmentosa. The long-term goal of this project is to determine the intracellular, extracellular, and environmental factors that influence clearance of all-trans retinal and protect photoreceptors from its toxicity.